Immune tolerance is highly relevant to a wide range of clinically important applications. Antigen-specific tolerance induction is a major goal for the treatment or prevention of autoimmune disease and graft rejection, which are currently controlled by nonspecific, immunosuppressive therapies that result in increased rates of infections, cancers and drug-related pathology. Other applications of immune tolerance induction include allergies and asthma, bone marrow replacement and protein based therapeutics.
One of the first practical applications of molecular biology has been the ability to produce large quantities of rare biological agents, many of which have therapeutic activity. It has been found, however, that during administration of these agents, a patient can mount an immune response, leading to the production of antibodies that bind and interfere with the therapeutic activity as well as cause acute or chronic immunologic reactions. This problem is most significant for therapeutics that are proteins because proteins are complex antigens and in many cases, the patient is immunologically naive to the antigens.
This type of immune response has been found in at least some patients with deficiency disorders such as hemophilia A (Aledort (1994) Am. J. Hemat. 47:208-217), diabetes mellitus (Gossain et al. (1985) Ann. Allergy 55:116-118), adenosine deaminase deficiency (Chaffee et al. (1993) J. Clin. Inv. 89:1643-1651), Gaucher disease (Richards et al. (1993) Blood 82:1402-1409), and Pompe disease (Amalfitano (2001) Genet Med 3:132-138. In hemophilia A, antibodies can inhibit factor VIII function requiring alternative treatment with activated prothrombin complex concentrates. In adenosine deaminase deficiency, antibodies to PEG modified adenosine deaminase enhance the clearance of the enzyme and lower its efficacy. In Gaucher, the induction of IgG1 antibodies has been associated with anaphylactoid reactions due to complement activation during infusions. In Pompe disease, replacement therapy with recombinant alpha-glucosidase resulted in the induction of antibodies in two of three patients treated, which resulted in declining efficacy of the therapy.
Similar immune responses have been found in the delivery of protein therapeutics by gene therapy. For example, viral proteins associated with vectors are targets of an immune response that can cause inflammation, shortened expression and prevented repeat administration of vector (Wilson and Kay (1995) Nature Med. 1:887-889). Antibody responses to the therapeutic protein have also been observed in gene therapy experiments and are part of an overall immune response that prevents long-term expression (Shull et al. (1996) Blood 88:377-379). Generalized immune suppression, blockade and immune deviation from humoral to cellular responses have been utilized to address this problem, but are not likely to ensure long lasting tolerance to the antigen.
Tolerance may be defined as the absence of an immune response to a specific antigen in the setting of an otherwise normal immune system. This state of specific immunological tolerance to self-components involves both central and peripheral mechanisms. Central tolerance (negative selection) is a consequence of immature T cells receiving strong intracellular signaling while still resident in the thymus, resulting in clonal deletion of autoreactive cells. Peripheral tolerance occurs when the immune system becomes unreactive to an antigen presented in the periphery, where, in contrast to the thymus, T cells are assumed to be functionally mature. Peripheral tolerance has been proposed to be the result of various mechanisms, including the development of antigen specific suppressor cells or other means of active tolerance, clonal deletion, and anergy. Autoreactive cells may be physically deleted by the induction of apoptosis after recognition of tolerizing antigen, may become anergic without deletion, or may be functionally inhibited by regulatory cytokines or cells. Although much has been learned in recent years, it is still difficult to predict the outcome of antigenic exposure in vivo, and further elucidation of tolerance mechanisms is needed at the basic level.
Numerous strategies have been developed to induce antigen specific tolerance in animal models, for example with respect to autoimmune disorders, such as multiple sclerosis (or experimental allergic encephalitis, EAE) or diabetes, as well as to prevent rejection of allogeneic tissue transplants. The major methods developed in mouse and rat models involve administration of high doses of soluble antigen, oral ingestion of antigens or intrathymic injection. The efficacy of these methods depends to varying degrees on clonal deletion, clonal anergy, active suppression by antigen-specific T cells and immune deviation from cellular to humoral immune responses.
Although administration of large quantities of soluble antigens has long been known to induce non-responsiveness to subsequent immunological challenge, studies of EAE have also highlighted the difficulties in this approach. The high doses required and the inconsistency of tolerance versus immune deviation make soluble antigen administration alone impractical for most gene therapy situations.
Administration of very large doses of antigen orally to mice has also been demonstrated to induce tolerance to protein antigens. However, extraordinary doses are required, and the results are complex. Certain doses are found to result in anergy, while other doses induce a form of antigen-specific bystander suppression, characterized by antigen-specific TH2 type responses. In addition, oral antigen can sensitize the immune system and lead to more severe disease.
Intrathymic injection of antigens or cells has been widely explored as a technique to induce central immune tolerance. Antigens injected and presented within the thymus can cause apoptosis or anergy of CD4+, CD8+ T cells in a process that may take up to 10 days. Like oral and soluble antigen tolerance, the tolerizing effect is dose dependent: low doses of antigen are sensitizing or provide only partial protection, whereas larger doses of antigen are tolerizing. The context of antigen presentation within the thymus is also important. Tolerance is most efficiently induced when the antigens are presented by host antigen presenting cells (APC). Once tolerized, the continued presence of antigen within the animal is needed for maintenance of tolerance, and depletion of mature T cells may also be required.
One strategy for tolerance induction is based on the discovery that optimal T cell activation requires both antigen-specific signals and non-antigen-specific signals. During antigen presentation, a variety of important bidirectional cognate interactions take place, with signaling to both the T cell and the antigen presenting cell. The best understood costimulatory signal is provided through the T cell surface molecule CD28. CD28 has two ligands, the homologous molecules CD80 (B7-1) and CD86 (B7-2); both are expressed on activated APC and some other cell types. Another pathway that has received significant attention and is important in T cell costimulation is that mediated by CD40 and its ligand CD154. CD154 is expressed on activated T cells, primarily CD4+ T cells.
In the past several years, a variety of laboratories have shown that blockade of T cell costimulatory signals can improve long-term allograft survival rates and induce transplantation tolerance. Most of these studies have used either CTLA4Ig, a fusion protein of CTLA-4 and human Ig that competitively binds CD80 and CD86, or a blocking monoclonal antibody to CD154. Costimulatory blockade has been partially successful in mouse and rat models of cardiac, hepatic, islet, renal, lung, and bone marrow transplantation. Although a single agent alone such as CTLA4Ig or anti-CD154 antibody can improve long-term graft survival rates, these agents by themselves are unlikely to yield indefinite graft survival; late allograft loss resulting from chronic rejection is the rule. Most commonly, either a transfusion of donor-specific lymphocytes or the combination of CTLA4Ig and anti-CD154 is required for long-term survival, with or without tolerance. Furthermore, the results in nonhuman primates are not as good as those in rodent models.
In murine models in which CTLA4Ig and/or anti-CD40 antibody has been used to induce tolerance, it has been shown that concomitant administration of cyclosporine prevents tolerance induction. It seems that the induction of tolerance in T cells deprived of costimulatory signals is an active process involving TCR signaling events that are sensitive to cyclosporine. Therefore, in the presence of cyclosporine, tolerance cannot be achieved by this means. These references therefore teach away from the use of cyclosporine in tolerance induction regimens.
In a protocol in which the combination of CTLA4Ig given 2 d after transplantation and donor-specific lymphocytes is used to induce cardiac allograft tolerance in mice, blockade of CTLA-4 at the time of transplantation prevents tolerance induction and leads to early rejection. Therefore, early CTLA-4 signals may be permissive for some T cell toleragenic/inhibitory strategies; without these signals, it may prove difficult to turn off the immune response. Similarly, in murine models of autoimmune disease, blockade of CTLA-4 exacerbates the duration and severity of the illness. Because agents such as CTLA4Ig prevent CD80 and CD86 from binding to both CD28 and CTLA-4, they have both the potential to block positive signals (through CD28) and the undesired ability to block negative signals (through CTLA-4).
In all of these methods, the tolerance is either unreliably induced, has not been achieved in humans or is not therapeutically or clinically useful. There is a clinical need for methods of preventing immune responses to antigens. The present invention addresses this problem.